1. Field of the Invention
This invention relates to four novel strains of Bacillus thuringiensis (the xe2x80x9cBTS02617A strainxe2x80x9d, the xe2x80x9cBTS02618A strainxe2x80x9d, the xe2x80x9cBTS02654B strainxe2x80x9d and the xe2x80x9cBTS02652E strainxe2x80x9d), each of which produces crystallized proteins (the xe2x80x9cBTS02617A crystal proteinsxe2x80x9d, the xe2x80x9cBTS02618A crystal proteinsxe2x80x9d, the xe2x80x9cBTS02654B crystal proteinsxe2x80x9d and the xe2x80x9cBTS02652E crystal proteinsxe2x80x9d, respectively) which are packaged in crystals (the xe2x80x9cBTS02617A crystalsxe2x80x9d, the xe2x80x9cBTS02618A crystalsxe2x80x9d, the xe2x80x9cBTS02654B crystalsxe2x80x9d and the xe2x80x9cBTS02652E crystalsxe2x80x9d, respectively) during sporulation. The BTS02617A, BTS02618A, BTS02654B and BTS02652E strains were deposited under the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganismsxe2x80x94Collection Laboratorium voor Microbiologie Belgium (xe2x80x9cBCCM-LMGxe2x80x9d), R. U. G., K. Ledeganckstraat 35, B-9000 Gent.
This invention also relates to an insecticide composition that is active against Lepidoptera and that comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, as such, or preferably the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals, crystal proteins or the active component(s) thereof as an active ingredient.
This invention further relates to a gene (the xe2x80x9cbTS02618A genexe2x80x9d), which is present in the genome of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains and which encodes an insecticidal protein (the xe2x80x9cBTS02618A protoxinxe2x80x9d) that is found in the BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals. The BTS02618A protoxin is the protein that is produced by the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains before being packaged into their respective BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals.
This invention still further relates to a toxin (the xe2x80x9cBTS02618A toxinxe2x80x9d) which can be obtained (e.g., by trypsin digestion) from the BTS02618A protoxin. The BTS02618A toxin is an insecticidally active protein which can be liberated from the BTS02617A crystals, the BTS02618A crystals, the BTS02654B crystals, and the BTS02652E crystals, which are produced by the BTS02617A strain, the BTS02618A strain, the BTS02654B strain and the BTS02652E strain, respectively. This toxin and its protoxin have a high activity against a wide range of lepidopteran insects, particularly against Noctuidae, especially against Spodoptera and Agrotis spp., but also against other important lepidopteran insects such as Pyralidae, particularly the European corn borer, Ostrinia nubilalis, Gelechiidae such as Phthorimaea operculella and Yponomeutidae such as Plutella xylostella. Furthermore, the BTS02618A protein is the first Bt protein with significant activity towards Agrotis segetum. This new characteristic of the BTS02618A protoxin and toxin (xe2x80x9c(pro)toxinxe2x80x9d), i.e., the combination of activity against different economically important Lepidopteran insect families such as Noctuidae, Yponomeutidae, Gelechiidae and Pyralidae, makes this (pro)toxin an ideally suited compound for combatting a wide range of insect pests by contacting these insects with the (pro)toxin, e.g., by spraying or by expressing the bTS02618A gene in plant-associated bacteria or in plants. The BTS02618A toxin is believed to represent the smallest portion of the BTS02618A protoxin which is insecticidally effective against Lepidoptera.
This invention also relates to transformed Bacillus thuringiensis strains, containing DNA sequences encoding a BTS02618A protein or variants thereof having substantially the same insecticidal activity.
This invention yet further relates to a chimeric gene that can be used to transform a plant cell and that contains the following operably linked DNA fragments:
1) a part of the bTS02618A gene (the xe2x80x9cinsecticidally effective bTS02618A gene partxe2x80x9d) encoding an insecticidally effective portion of the BTS02618A protoxin, preferably a truncated part of the bTS02618A gene (the xe2x80x9ctruncated bTS02618A genexe2x80x9d) encoding just the BTS02618A toxin;
2) a promoter suitable for transcription of the insecticidally effective bTS02618A gene part in a plant cell; and
3) suitable 3xe2x80x2 end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618A gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the xe2x80x9cbTS02618A chimeric genexe2x80x9d.
This invention also relates to:
1) a cell (the xe2x80x9ctransformed plant cellxe2x80x9d) of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene; and
2) a plant (the xe2x80x9ctransformed plantxe2x80x9d) which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene, and which is resistant to Lepidoptera.
This invention still further relates to:
1) a microbial organism, such as B. thuringiensis or Pseudomonas spp., the genome of which is transformed with all or part of the bTS02618A gene; and
2) a microbial spore, containing a genome which is transformed with all or parts of the bTS02618A gene.
Another embodiment of the present invention relates to artificially made bTS02618A genes which encode BTS02618A proteins, and to proteins which are more protease resistant than native Bt proteins, more preferably the native BTS02618A protein. A particular example of a protein that is more protease resistant is the BTS02618Aa protein. Furthermore, the present invention also relates to a DNA sequence encoding the BTS02618Aa protein.
Yet another embodiment of the present invention relates to a chimeric gene that can be used to transform a plant cell and that contains:
1) a DNA sequence encoding an insecticidally effective portion of the BTS02618Aa protoxin, preferably a truncated part of the bTS02618Aa gene (the xe2x80x9ctruncated bTS02618Aa genexe2x80x9d) encoding just the BTS02618Aa toxin;
2) a promoter suitable for transcription of the insecticidally effective bTS02618Aa gene part in a plant cell; and
3) suitable 3xe2x80x2 end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618Aa gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the xe2x80x9cbTS02618Aa chimeric genexe2x80x9d.
This invention further relates to:
1) a cell (the xe2x80x9ctransformed plant cellxe2x80x9d) of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618Aa gene part, preferably the bTS02618Aa chimeric gene; and
2) a plant (the xe2x80x9ctransformed plantxe2x80x9d) which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618Aa gene part, preferably the bTS02618Aa chimeric gene, and which is resistant to Lepidoptera.
This invention still further relates to:
1) a microbial organism, such as B. thuringiensis or Pseudomonas spp., the genome of which is transformed with all or part of a DNA sequence encoding the BTS02618Aa protein; and
2) a microbial spore, containing a genome which is transformed with all or part of the bTS02618Aa gene.
Yet another embodiment of the present invention relates to insecticidal compositions that are active against Lepidoptera and that comprise a more protease resistant Bt protein, more particularly the BTS02618Aa protein or a variant thereof which has substantially the same insecticidal activity.
2. Description of the Prior Art
B. thuringiensis (xe2x80x9cBtxe2x80x9d) is a Gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae. These crystal proteins and corresponding genes have been classified based on their structure and insecticidal spectrum (H{dot over (o)}fte and Whiteley, 1989). The four major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), and Diptera-specific (cryIV) genes.
The fact that conventional submerged fermentation techniques can be used to produce Bt spores on a large scale makes Bt bacteria commercially attractive as a source of insecticidal compositions.
Gene fragments from some Bt strains, encoding insecticidal proteins, have heretofore been identified and integrated into plant genomes in order to render the plants insect-resistant. However, obtaining expression of such Bt gene fragments in plants is not a straightforward process. In order to achieve optimal expression of an insecticidal protein in plant cells, it has been found necessary to engineer each Bt gene fragment in a specific way so that it encodes a part of a Bt protoxin that retains substantial toxicity against its target insects (European patent application (xe2x80x9cEPAxe2x80x9d) 86/300,291.1 and 88/402,115.5; U.S. patent application 821,582, filed Jan. 22, 1986).
In accordance with this invention, four novel Bt strains, i.e., the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains, are provided. The BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals and crystal proteins, the BTS02618A protoxin and toxin produced by the strains during sporulation, and insecticidally effective portions of the BTS02618A protoxin, as well as equivalents of these crystals, crystal proteins, protoxin, toxin and insecticidally effective protoxin portions, each possess insecticidal activity and can therefore be formulated into insecticidal compositions against Lepidoptera in general, and particularly against Noctuidae, such as Agrotis spp. (cutworms such as Agrotis ipsilon and Agrotis segetum), Mamestra spp. (e.g., the cabbage moth, Mamestra brassica) and Spodoptera spp. (armyworms, such as Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis and Spodoptera litura), against Pyralidae (e.g., the European corn borer, Ostrinia nubilalis), against Gelechiidae such as Phthorimaea operculella and Yponomeutidae (such as Plutella xylostella) which are major pests of various economically important crops, such as corn, cotton and many vegetables such as Brassicas.
Also in accordance with this invention, a plant cell genome is transformed with the insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, or an equivalent thereof such as a modified, synthetic bTS02618A gene. It is preferred that this transformation be carried out with the bTS02618A chimeric gene. The resulting transformed plant cell can be used to produce transformed plants, seeds of transformed plants and plant cell cultures consisting essentially of the transformed cells. The transformed cells in some or all of the tissues of the transformed plants: 1) contain the insecticidally effective bTS02618A gene part as a stable insert in their genome, and 2) express the insecticidally effective bTS02618A gene part by producing an insecticidally effective portion of its BTS02618A protoxin, preferably its BTS02618A toxin, thereby rendering the plant resistant to Lepidoptera. The transformed plant cells of this invention can also be used to produce, for recovery, such insecticidal Bt proteins.
Further in accordance with this invention, a process is provided for rendering a plant resistant to Lepidoptera by transforming the plant cell genome with the insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, or an equivalent thereof. In this regard, it is preferred that the plant cell be transformed with the bTS02618A chimeric gene.
Yet further in accordance with this invention, there are provided the BTS02618A protoxin, the insecticidally effective portions of such protoxin and the BTS02618A toxin, as well as functional parts of the BTS02618A toxin, as well as the bTS02618A gene, the insecticidally effective bTS02618A gene part, the truncated bTS02618A gene and the chimeric bTS02618A gene, as well as their equivalents.
Also in accordance with this invention, a DNA sequence, either natural or artificial, encoding the BTS02618A protoxin or insecticidally effective portions thereof, such as the toxin, is provided.
Also in accordance with this invention are provided an insecticidal composition against Lepidoptera, particularly Noctuidae, Pyralidae, Gelechiidae and Yponomeutidae, and a method for controlling Lepidoptera, particularly Noctuidae, Pyralidae, Gelechiidae and Yponomeutidae, with the insecticidal composition, wherein the insecticidal composition comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, crystals and/or crystal proteins or the BTS02618A protoxin, toxin and/or insecticidally effective protoxin portions or their equivalents.
Also in accordance with this invention, bacteria, particularly E. coli and Bacillus thuringiensis, transformed to express a DNA encoding the BTS02618A protein variant, such as the BTS02618Aa protein or more improved protease resistant Bt proteins are provided.
Furthermore, in accordance with this invention, an artificial DNA sequence encoding the BTS02618A protein, as well as new forms of Bt proteins with improved protease resistance, more particularly the BTS02618Aa or the modified BTS02618A protein are provided, and DNA sequences encoding these new proteins. Further provided are plant cells expressing an artificial DNA sequence encoding the BTS02618A toxin or Bt toxins with improved protease resistance, more preferably the BTS02618Aa toxin.
Also provided is an insecticidal composition, comprising as an active ingredient the BTS02618Aa protein, or a variant thereof with substantially the same insecticidal activity. Also provided is a method to combat Lepidopteran insects by contacting these insects with Bt proteins having improved protease resistance, more preferably the BTS02618Aa protein or a variant thereof.
More specifically provided are new Bt proteins, preferably Lepidoptera active Bt proteins, having substantially the same insecticidal activity as the native Bt protein, but characterized in their resistance to further proteolytic cleavage of the about 60 to 70 kD toxin form. Such new Bt proteins have inactivated internal protease cleavage sites, so that these proteins have increased stability while retaining substantially the same insecticidal activity. Thus, these new Bt proteins are not readily cleaved into smaller proteolytic fragments which lower their insecticidal activity upon prolonged incubation in the presence of proteases.